Acute respiratory infection with mouse adenovirus type 1.

Abstract

Studies of the pathogenesis of adenovirus respiratory disease are limited by the strict species-specificity of the adenoviruses. Following intranasal inoculation of adult C57BL/6 mice with mouse adenovirus type 1 (MAV-1), we detected MAV-1 early region 3 (E3) and hexon gene expression in the lungs at 7 days post-infection (dpi). We detected MAV-1 E3 protein in the respiratory epithelium at 7 dpi. We did not detect viral mRNA or protein at 14 dpi, but MAV-1 DNA was detected by PCR at 21 dpi. Chemokine transcript levels increased between 7 and 14 dpi in the lungs of infected mice. MAV-1 infection induced a patchy cellular infiltrate in lungs at 7 and 14 dpi. This is the first report demonstrating the presence of MAV-1 in the respiratory epithelium of infected mice and describing chemokine responses in the lung induced by MAV-1 respiratory infection. MAV-1 infection of mice has the potential to serve as a model for inflammatory changes seen in human adenovirus respiratory disease.

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Acute respiratory infection with mouse adenovirus type 1
Jason B. Weinberga,*, Gregory S. Stempflea, John E. Wilkinsonb, John G. Youngerc, and
Katherine R. Spindlerd
a University of Michigan Health System, Division of Pediatric Infectious Diseases, Department of Pediatrics,
L2225 Women’s/0244, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0244, USA
b University of Michigan, Department of Pathology and Unit for Laboratory Animal Medicine, 018 ARF,
1500 West Medical Center Drive, Ann Arbor, MI 48109-0614, USA
c University of Michigan, Department of Emergency Medicine, B1354 Taubman Center, 1500 East Medical
Center Drive, Ann Arbor, MI 48109-0303, USA
d University of Michigan, Department of Microbiology and Immunology, 6724 Medical Sciences Building II,
1150 West Medical Center Drive, Ann Arbor, MI 48109-0620, USA
Abstract
Studies of the pathogenesis of adenovirus respiratory disease are limited by the strict species-
specificity of the adenoviruses. Following intranasal inoculation of adult C57BL/6 mice with mouse
adenovirus type 1 (MAV-1), we detected MAV-1 early region 3 (E3) and hexon gene expression in
the lungs at 7 days post-infection (dpi). We detected MAV-1 E3 protein in the respiratory epithelium
7 dpi. We did not detect viral mRNA or protein at 14 dpi, but MAV-1 DNA was detected by PCR
at 21 dpi. Chemokine transcript levels increased between 7 and 14 dpi in the lungs of infected mice.
MAV-1 infection induced a patchy cellular infiltrate in lungs at 7 and 14 dpi. This is the first report
demonstrating the presence of MAV-1 in the respiratory epithelium of infected mice and describing
chemokine responses in the lung induced by MAV-1 respiratory infection. MAV-1 infection of mice
has the potential to serve as a model for inflammatory changes seen in human adenovirus respiratory
disease.
Keywords
Adenovirus; Chemokine; Lung; Inflammation
Introduction
Human adenoviruses cause a wide range of upper and lower respiratory tract infections in
children (Carballal et al., 2002; Edwards et al., 1985; Larranaga et al., 2000; Pacini et al.,
1987). Adenoviruses are also a common cause of upper respiratory tract infection and
pneumonia in military recruits (Huebner et al., 1958; Kolavic-Gray et al., 2002; Mogabgab,
1968). Immunocompromised patients experience greater problems due to adenovirus
infections, including more aggressive respiratory infections, transplant loss and death
(Kojaoghlanian et al., 2003). A potential long-term consequence of persistent adenovirus
infection is an increased risk for the development of asthma and chronic obstructive pulmonary
disease (COPD) (Hogg, 1999, 2001). Human adenoviruses are also significant as vectors for
in vitro and in vivo delivery of genes into mammalian cells (reviewed in Kay et al., 2001).
* Corresponding author. Fax: +1 734 936 7635..
E-mail addresses:jbwein@umich.edu (J.B. Weinberg),stempfle@umich.edu (G.S. Stempfle),jerby@umich.edu (J.E.
Wilkinson),jyounger@umich.edu (J.G. Younger),krspin@umich.edu (K.R. Spindler).
NIH Public Access
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Published in final edited form as:
Virology. 2005 September 30; 340(2): 245–254.
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Chemokines are chemotactic cytokines that serve as chemoattractants for cells involved in the
inflammatory response to stimuli such as infection (Luster, 1998; Rollins, 1997; Taub, 1996).
Combined in vitro, ex vivo and in vivo data suggest that both intact (Alcorn et al., 2001; Booth
et al., 2004; Harrod et al., 1998; Kajon et al., 2003; Leland Booth and Metcalf, 1999; Otake et
al., 1998) and replication-deficient (Amin et al., 1995; Kodama et al., 2001; Noah et al.,
1996; Schwarz et al., 1999) human adenoviruses are capable of inducing chemokine responses
in human, murine and bovine lung. Chemokines potentially play a significant role in the
pathophysiology of asthma (Blease et al., 2000; Lukacs, 2001) and COPD (Beeh et al., 2003;
Qiu et al., 2003; Saetta et al., 2002; Traves et al., 2002). Chemokine responses to adenovirus
vectors contribute to difficulties encountered with limited persistence of the vector in its host,
low levels of expression of desired genes and potential morbidity associated with use of the
vector (reviewed in Liu and Muruve, 2003).
A variety of models have been used to study chemokine responses to adenovirus infection. In
vitro systems have used infection of cell lines with adenovirus or transduction with adenovirus
vectors (Alcorn et al., 2001; Kodama et al., 2001; Leland Booth and Metcalf, 1999; Schwarz
et al., 1999). An ex vivo lung slice model has been used to examine chemokine responses to
adenovirus infection in the context of a relevant tissue (Booth et al., 2004). The majority of
these studies have focused on upregulation of interleukin 8 (IL-8), a CXC chemokine that
serves as a major neutrophil chemoattractant and activator (Baggiolini et al., 1994). Relatively
few studies have used in vivo animal models to examine chemokine responses to respiratory
infection with human adenovirus (Harrod et al., 1998; Kajon et al., 2003). In vivo studies of
human adenovirus pathogenesis are limited by the strict species-specificities of the
adenoviruses. Because exposure of mice to human adenovirus does not result in a fully
permissive infection (Ginsberg et al., 1991; Kajon et al., 2003), this system does not allow for
a complete assessment of host inflammatory responses to persistent adenovirus infection.
Human adenovirus infection of cotton rats (Sigmodon hispidus) has shown the most promise
as an animal model of human adenovirus respiratory disease (Pacini et al., 1984; Prince et al.,
1993), but it depends on infection with a high dose of a virus that is not fully adapted to the
host. This drawback is particularly relevant in the study of links between adenovirus infection
and chronic lung disease such as asthma or COPD, which are more likely to be associated with
effects of persistent rather than acute infection.
Mouse adenovirus type 1 (MAV-1) serves as an excellent animal model system for studying
adenovirus pathogenesis, providing the means to define viral and host factors involved in both
acute and persistent adenovirus infections (reviewed in Smith and Spindler, 1999). The MAV-1
virion is structurally similar to that of human adenoviruses (Wigand et al., 1977), and its
genomic organization is also similar to that of human adenovirus (Meissner et al., 1997). With
the exception of the early region 3 (E3) gene (Beard et al., 1990), MAV-1 gene products have
sequence similarity to those of human adenoviruses (Ball et al., 1988, 1989, 1991; Beard et
al., 1990; Cauthen and Spindler, 1996; Kring et al., 1992; Kring and Spindler, 1990). Of
particular interest, given the potential association between the human adenovirus early region
1A (E1A) gene and long-term sequelae of infection such as COPD (Elliott et al., 1995; Matsuse
et al., 1992), MAV-1 E1A has approximately 40% similarity to human adenovirus E1A, with
the strongest similarity in conserved region 2 (CR2) (Ball et al., 1988). MAV-1 E1A shares
functional roles of human adenovirus E1A, binding to the mouse cellular proteins,
retinoblastoma protein and pRb-related protein (Fang et al., 2004; Smith et al., 1996) and
transactivating the human adenovirus type 5 E3 promoter (Ball et al., 1988).
Few studies directly address respiratory infection with MAV-1 or the role of chemokine
responses to MAV-1. Following intraperitoneal (i.p.) MAV-1 inoculation of adult C57BL/6
mice, increased mRNA levels for the chemokines IP-10, MCP-1 and TCA-3 were detected in
the brain between 3 and 4 days post-infection (dpi) (Charles et al., 1999). MAV-1 organ tropism
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following intranasal (i.n.) inoculation of outbred mice is similar to that observed following i.p.
inoculation (Kajon et al., 1998). In each case, virus was identified in multiple organs including
lung, spleen and brain (Kajon et al., 1998). Following i.n. inoculation of newborn BALB/c
mice less than 24 h old, peribronchiolar infiltrates comprised of macrophages, and lymphocytes
were noted 3 dpi (Gottlieb and Villarreal, 2000).
We and others have used mouse models to study the chemokine responses to respiratory
infections with a variety of viruses, including acute viral infections with respiratory syncytial
virus (Domachowske et al., 2000; Haeberle et al., 2001; John et al., 2003; Mejias et al.,
2004; Miller et al., 2004; Power et al., 2001; Tekkanat et al., 2002) and influenza virus (Dawson
et al., 2000; Tumpey et al., 2000) as well as persistent viral infections such as those caused by
murine gammaherpesvirus 68 (Sarawar et al., 2002; Weinberg et al., 2002, 2004). In this study,
we demonstrate that respiratory infection is established following i.n. inoculation of C57BL/
6 mice with MAV-1. Respiratory infection with MAV-1 results in chemokine upregulation
and cellular inflammation in the lung. These data indicate that respiratory infection with
MAV-1 may serve as a useful model to study adenovirus respiratory disease.
Results
Clinical signs of MAV-1 infection
In order to study MAV-1 respiratory infection, we inoculated 4- to 6-week-old C57BL/6 mice
i.n. with 105 plaque-forming units (PFU) of MAV-1 and carefully monitored them until 14 dpi.
At no time point did mice infected with MAV-1 exhibit clinical signs suggesting respiratory
distress such as tachypnea or labored respirations. CNS symptoms such as ataxia,
hyperreflexia, hyper-esthesia, paresis and flaccid paralysis were absent in all infected mice.
Detection of MAV-1 in the lung
Using in situ hybridization, Kajon et al. (1998) demonstrated the presence of MAV-1 in the
lungs of adult outbred mice following both i.n. and i.p. inoculation. We sought to confirm and
expand on these findings in the current study using inbred mice. In three separate experiments
(n = 2 to 6 mice per condition at each time point), we harvested total RNA from lungs of mice
at 1, 4, 7 and 14 dpi. We used ribonuclease protection assay (RPA) to detect the expression of
MAV-1 E3 and hexon genes in the lung at each time point. We did not detect E3 gene expression
at 1 or 4 dpi (data not shown). We detected E3 gene expression in 4 of 6 mice at 7 dpi (Fig.
1A) but did not detect E3 gene expression in any mice at 14 dpi. Expression of the late gene
encoding the structural protein, hexon, was also detected at 7 dpi but not at 14 dpi in the lungs
of infected mice (Fig. 1A). Paralleling these data, we found evidence of infectious virus in lung
homogenates at 7 dpi but not at 14 dpi by plaque assay titration on mouse 3T6 fibroblast
monolayers (data not shown). As expected, we did not detect E3 or hexon gene expression in
the lungs of mock-infected animals at any time point (Fig. 1A).
MAV-1 has a tropism for endothelial cells in the CNS and other organs (Charles et al., 1998;
Guida et al., 1995; Kajon et al., 1998; Moore et al., 2003). To determine whether MAV-1 is
capable of infecting respiratory epithelial cells following i.n. inoculation, we performed
immunohistochemistry on paraffin-embedded sections of lungs harvested at various time
points following infection to localize MAV-1 E3 protein. At 7 dpi, virus-specific staining was
evident in respiratory epithelial cells lining the medium and large airways of the lung (Fig. 2).
At 14 dpi, MAV-1-specific staining was not present in respiratory epithelial cells of infected
lungs, although there was occasional staining of vascular endothelial cells in the lung (arrow,
Fig. 2). No staining was noted using preimmune rabbit serum as a primary antibody (data not
shown).
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Expression of hexon and E3 genes was not detected in lungs harvested from a separate group
of mice at 21 dpi (data not shown). However, we detected the presence of MAV-1 DNA in the
lungs of 2 of 3 mice at 21 dpi by PCR (Fig. 1B). Together, these data suggest that i.n. inoculation
with MAV-1 resulted in a productive pulmonary infection in C57BL/6 mice involving
respiratory epithelial cells. Following acute infection, MAV-1 genome persisted in the lung in
the absence of detectable viral gene expression.
Chemokine responses to MAV-1 infection
Chemokine upregulation induced by MAV-1 has been described in the CNS but not the lung
following i.p. inoculation (Charles et al., 1999). We used RPA to quantify chemokine gene
expression in the lungs of mice infected i.n. with MAV-1. Data from a representative
experiment are presented in Fig. 3. Prior to infection or at 1 and 4 dpi, we noted little chemokine
expression in the lungs of mice, and levels of chemokine expression did not differ between
MAV-1-infected and mock-infected mice (data not shown). In contrast, MAV-1 infection
upregulated the expression of nearly all chemokine genes measured at later time points relative
to mock infection (Fig. 3A). Levels of expression of IP-10 and Ltn peaked at 7 dpi, while levels
of expression of MIP-1α, MIP-1β and RANTES peaked at 14 dpi. MCP-1 and TCA-3
expression was increased in MAV-1-infected animals to similar levels at 7 and 14 dpi. No
significant differences in MIP-2 expression between MAV-1-infected and mock-infected mice
were detected at any time point. Expression of the eotaxin gene was not reliably detected by
RPA in lungs of MAV-1-infected or mock-infected mice at any time point (data not shown).
Similar RPA results were obtained in separate experiments from infected mice at identical time
points (data not shown).
To verify that altered levels of chemokine gene expression after infection were reflected in
chemokine protein levels, we measured RANTES protein levels in lung homogenates at 7 and
14 dpi (Fig. 3B). At 7 dpi, levels of RANTES protein were not increased in the lungs of infected
compared to mock-infected mice. At 14 dpi, when RANTES gene expression was dramatically
upregulated in the lungs of infected mice (Fig. 3A), levels of RANTES protein were also
significantly elevated compared to mock-infected mice (Fig. 3B).
Cellular inflammatory responses to MAV-1 infection in the lung
Chemokines are chemotactic cytokines that form a concentration gradient to attract cells
involved in the inflammatory response to pathogens and other stimuli (Luster, 1998). Based
on the chemokine upregulation we detected in the lungs and brains of infected mice, we
predicted that a cellular inflammatory response would accompany MAV-1 respiratory
infection. To assess cellular inflammation, we examined hematoxylin and eosin-stained
sections of lungs and brain from MAV-1-infected and mock-infected mice. No cellular
infiltrate was present in the lungs of mock-infected mice at any time point (Fig. 4A) or in the
lungs of infected mice at 1 and 4 dpi (data not shown). A mild patchy interstitial pneumonitis
was present in lungs of infected mice at both 7 and 14 dpi (Fig. 4A) that was characterized by
a predominantly mononuclear infiltrate and thickened alveolar walls. In addition, scattered
areas of hypercellularity were focused around medium and large airways in the lungs of infected
mice at 7 dpi; these areas consisted predominantly of mononuclear cell infiltrates. These
changes were also seen, though to a lesser extent, at 14 dpi. Thus, maximum cellular
inflammation in the lung was present at 7 dpi when virus was detected in the lung by RPA,
immunohistochemistry and plaque assay and was reduced when virus had been cleared from
the lung at 14 dpi.
In a separate experiment, we used bronchoalveolar lavage (BAL) to characterize the airway
cellular inflammatory response to MAV-1 respiratory infection at 7 dpi, when the greatest
amount of cellular inflammation was noted in the lungs (Fig. 4B). BAL fluid from mock-
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infected animals had a monotonous population of alveolar macrophages. These cells were also
evident in BAL fluid from MAV-1-infected animals. In addition, BAL fluid from infected
animals contained a variable degree of neutrophils (open arrow, Fig. 4B), ranging from 1.5%
to 28.3% of the total population of cells (Fig. 4C). In contrast, BAL fluid from mock-infected
animals contained only between 0.3% and 0.5% neutrophils. Only rare leukocytes of other
types were noted in either mock-infected or MAV-1-infected animals, and their percentages
did not differ among groups (data not shown).
Discussion
We demonstrate here that MAV-1 is capable of establishing a respiratory infection in the lungs
of C57BL/6 mice and describe the novel finding that MAV-1 protein is present in respiratory
epithelial cells following i.n. inoculation. We observed a parallel between viral gene expression
and chemokine upregulation in the lungs. As expected from the chemokine upregulation we
detected, MAV-1 infection in the lung elicited a cellular inflammatory response that resulted
in an interstitial pneumonitis and foci of inflammation around larger airways.
Previous work from our laboratory showed that MAV-1 DNA could be detected using in situ
hybridization in the lung following both i.n. and i.p. inoculation of NIH Swiss outbred mice
with MAV-1, but its distribution was limited to the vascular endothelium (Kajon et al.,
1998). Here, we found MAV-1 protein in both vascular endothelium and respiratory epithelium
following i.n. inoculation of C57BL/6 mice. Several possibilities may explain the apparent
difference in cell tropism. The lack of detection of MAV-1 nucleic acid in the earlier report is
possibly due to a lower viral dose, 103 PFU, versus 105 PFU used here. The higher dose may
increase the efficiency of entry into a cell. Following virus entry, the higher dose may result
in increased viral replication to levels whereby viral protein is detectable by
immunohistochemistry. We believe that a less likely explanation is that detection of protein in
respiratory epithelial cells using immunohistochemistry was more sensitive than detection of
MAV-1 DNA by in situ hybridization.
Another possibility explaining the apparent cell tropism difference between this study and the
previous study (Kajon et al., 1998) is that different mouse strains were used, C57BL/6 and
NIH Swiss outbred mice, respectively. Differential host susceptibility to MAV-1 infection is
seen among various inbred and outbred mouse strains (Guida et al., 1995; Kring et al., 1995;
Spindler et al., 2001). The 50% lethal dose (LD50) for outbred NIH Swiss outbred mice is 17
PFU (Kring et al., 1995), much lower than the LD50 of >104.4 PFU for C57BL/6 mice (Spindler
et al., 2001). The underlying basis for these differences in susceptibility is unknown but is
likely to involve mouse strain-specific differences in immune responses to MAV-1. For
instance, chemokine responses to MAV-1 infection in the brain differ between C57BL/6 and
BALB/c mice (Charles et al., 1999). It is therefore possible that our ability to detect MAV-1
in the respiratory epithelium in the present study and not in our previous study (Kajon et al.,
1998) is based on differing abilities of the two mouse strains to efficiently prevent or control
viral infection in respiratory epithelial cells.
Histopathologic descriptions of adenovirus respiratory disease in humans are quite limited and
largely include descriptions of fatal disease and disease in immunocompromised individuals
(Becroft, 1967, 1971; Chany et al., 1958; Zahradnik et al., 1980). MAV-1-induced respiratory
disease in C57BL/6 mice appears to share some characteristics with human disease. The
respiratory epithelium appears to be involved in both cases, although the significant epithelial
damage seem in some human cases (Becroft, 1967, 1971; Chany et al., 1958) was not prominent
in this study. Histopathologic features of respiratory disease induced by MAV-1 are also similar
in some ways to those present in the cotton rat model of human adenovirus type 5 (Prince et
al., 1993), including the presence of viral antigen in respiratory epithelial cells and a cellular
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